Author: Emily Cowlishaw
Introduction
The collection of bacteria, fungi, viruses, and eukaryotes that colonize the human body, collectively known as the microbiota, has shaped human evolution. It is estimated that the human gut is populated by at least 1014 bacteria at any given time. The human body acts as a diverse ecosystem, harboring the usual commensal and symbiotic organisms, as well as the less frequent pathogenic microbes. Some functions of the gut microbiota include digestion of compounds the human body cannot process, production of vitamins, activating the anti-inflammatory immune response, and providing colonization resistance to prevent the growth of harmful bacteria. However, the use of antibiotics can greatly alter and decrease the diversity of the gut microbiota, leading to opportunistic infections.
Treatment options for bacterial infections are quickly diminishing as antimicrobial resistance is on a continued rise. The ever-decreasing efficacy of antibiotics can be attributed to many different factors including misuse, over-prescription, agricultural administration, and environmental acquisition of antibiotic resistance genes. Bacteria in the human gut share similar properties and demonstrate an ideal setting to transfer genetic elements. Horizontal gene transfer (HGT) of antibiotic resistance genes has been occurring since before the development and exploitation of antibiotics. The microfloras of the gut not only share antibiotic resistance genes among themselves, but also gather and contribute resistance genes to non-resident organisms that are just passing through the gastrointestinal (GI) tract.
Human Gut Microbiota Serves as Reservoir for ARG
Antibiotic resistance can arise from mutations or various mechanisms of HGT of antimicrobial resistance genes (ARG) between organisms. Conjugation is a method of HGT in which the two organisms participating in the genetic transfer form a pilus, a hollow bridge-like formation, that allows the flow of mobile genetic elements. Transductionrefers to bacteriophages gathering resistance genes from one organism and delivering them to another. Transformation is the uptake of naked DNA; transformation contributes only minimally to the exchange of ARG. Conjugation appears to be the most significant method of HGT of ARG as the cell-to-cell contact in the gut is extremely high. Treatment with antibiotics has been shown to significantly alter the microbiota and positively select for organisms that have mechanisms or genes of resistance, ultimately leading to an environment rich in ARG (see figure 1). Species which have not developed a mechanism of antibiotic resistance are eliminated, resulting in a decrease in bacterial diversity.
The commensal organisms that typically inhabit the gut function in preventing opportunistic pathogens from colonizing the GI tract cause colonization resistance. Colonization resistance can occur from commensal bacteria using up the nutrients in the GI tract, providing no energy source for opportunistic pathogens passing through. Some organisms of the gut have been shown to provide colonization resistance by producing bacteriocins, which are compounds that are active against other bacteria. Thus, antibiotic treatment may lead to loss of colonization resistance and allow for infection from pathogenic organisms such as Clostridium difficile, Listeria monocytogenes, and Vancomycin-resistant enterococci (VRE). In the gut microbiota, the two most abundant phyla are Bacteroidetesand Firmicutes. Various members of the Firmicutes phyla have been identified in transmission of vancomycin resistance genes to VRE, while Bacteroides are primarily responsible for harboring and transferring many ARG for clindamycin and tetracycline.
Firmicutes and ARG
The phylum Firmicutes contains more than 250 different bacterial species including the genera Streptococcus, Clostridium, Staphylococcus, Lactobacillus, and Enterococcus. Enterococcal species are known for their ability to cause nosocomial infections and have resistance to vancomycin; these bacteria are called vancomycin-resistant enterococci (VRE). In Australia, vanB is the most numerous gene associated with VRE. People of varying ages, with no history of VRE infections, were screened for presence of vanB gene in non-enterococcal organisms by fecal sample. In preschool aged children, 15 out of the 56 participants sampled were determined to have non-enterococcalvanB in fecal samples. In the adult population that was sampled, 57 of the 91 participants had vanB positive fecal samples. The population of adults on hemodialysis had 49 total participants, of which 22 tested positive for vanB in their fecal samples.
Bacteroides fragilis and ARG
Bacteroides are a commensal anaerobic bacterium that inhabits the human GI tract, accounting for approximately one quarter of all colonic bacteria. Species of Bacteroides, including B. thetaiotaomicron and B. ovatus, have been shown to play a key role in digesting plant glycans that human digestive enzymes are unable to utilize. It is estimated that microbes like these contribute up to 10% of the daily calorie uptake in humans. Some species of Bacteroides have also been known to be opportunistic pathogens, commonly found in abdominal abscesses, soft tissue infections, and surgical wounds. Examining a subset of 161 Bacteroides isolates (most commonly Bacteroides fragilis) from Europe between 2008 and 2009 for presence of antibiotic resistance genes depicted just how many ARG commensal bacterium the GI tract can harbor. In this study, the 161 samples were composed of 128 strains of B. fragilis, and the remaining non-fragilis Bacteroides (NFB) contained seven strains, including B. thetaiotaomicron, B. ovatus, and B. eggerthii. Real-time PCR was used to detect the presence of antibiotic resistance genes in these Bacteroides isolates.
To test for resistance to ampicillin, the cepA gene was recognized by RT-PCR; while all 128 strains of B. fragilis were resistant to ampicillin, the cepA gene was only identified in 101 of the 128 strains of B. fragilis. Similarly, in the NFB group, 33 strains were shown to be resistant to ampicillin; only 12 harbored the cepA gene. Resistance to cefoxitin was shown in eleven B. fragilis strains, three of which contained the cfxA gene. In the NFB, resistance was observed in nine strains, with only one having the cfxA gene. Conversely, when looking at imipenem resistance, only one B. fragilis strain was phenotypically resistant, but the imipenem resistance gene, cfiA, was identified in twelve strains. Clindamycin resistance was observed in 40 of the 161 isolates. The genes associated with clindamycin resistance were variably present within these 40 resistant strains; 30 harbored ermF; 14 contained linA; 11 had mefA; ermG and msrSA were present in 9; and one harbored ermB. Genes related to tetracycline resistance include tetQ, which was observed in 129 of the 161 strains; tetX, which was observed in 16 of the 161; and tetX1, which was recorded in 8 of the 161 strains. Of these genes recognized in Bacteroides, ermG, ermB, ermF, and tetQ have been identified in gene transfer events between Bacteroides and Gram-positive pathogens (see Figure 2).
Another example of ARG transfer among Bacteroides pertains to a plasmid identified in twelve B. fragilis clinical isolates that contributes to carbapenem resistance. The plasmid, pBFUK1, was shown to contain the genes cfiA, tnpA, tnpB, repA, mobA, mobB, and mobC variably among the twelve isolates; two strains of the twelve harbored all of these genes. The cifA gene is recognized as responsible for high resistance to class B metallo-β-lactamases, including carbapenem. The cifA gene is typically found on the chromosome, rather than mobilized on a plasmid. Of the other genes, tnpA and tnpB are transposases, repA is a replication protein, and mobA, mobB, and mobC are genes that encode mobilization proteins. All of the genes from all of the twelve strains showed almost 100% similarity to another commensal Bacteroides species, including the B. fragilis, B. intestinalis, and B. eggerthii, indicating the plasmid was likely acquired in the human gut. Carbapenem resistance is an enormous problem in multi-drug resistant infections as carbapenem is usually a last resort broad-spectrum antibiotic. Resistance to carbapenem has been observed in several superbugs, including Acinetobacter baumannii and Klebsiella pneumoniae.
Conclusions
The human microbiota serves many important functions that prevent colonization of pathogenic organisms. However, the abundance of organisms in the GI tract makes it an optimal place for HGT to occur. Antimicrobial resistance genes are transferred between commensal organisms of the microbiota, as well as organisms that are passing through. Identical ARG have been observed between commensal Gram-negative organisms, such as Bacteroides and Gram-positive organisms, as the result of HGT events. The combination of antibiotic abuse and transfer of ARG between commensals, symbionts, and pathogens in the human gut has greatly contributed to the development of antibiotic resistant microbes.
This article was prepared by the author in their personal capacity. The opinions expressed in this article are the author's own and do not reflect the view of their place of employment.
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